Prevalence of Beijing Central Asian/Russian Cluster 94-32 among Multidrug-Resistant M. tuberculosis in Kazakhstan

The Beijing genotype is the most distributed M. tuberculosis family in Kazakhstan. In this study, we identified dominant Beijing clusters in Kazakhstan and assessed their drug susceptibility profiles and association with the most widely spread mutation Ser531Leu of the rpoB gene and the mutation Ser315Thr of the katG gene associated with resistance to rifampicin and isoniazid, respectively. M. tuberculosis isolates (n = 540) from new TB cases were included in the study. MIRU-VNTR genotyping was performed for 540 clinical isolates to determine M. tuberculosis families using 24 loci. RD analysis was additionally performed for the Beijing isolates. The identification of mutations in the drug-resistance genes of M. tuberculosis was performed with allele-specific real-time PCR and Sanger sequencing. The Beijing genotype was identified in 60% (324/540) of the clinical isolates. Central Asian/Russian cluster 94-32 was the most distributed cluster among the Beijing isolates (50.3%; 163/324). Three other dominant Beijing clusters were identified as 94-33 (3.4%; 11/324), 100-32 (3.1%; 10/324) and 99-32 (3.1%; 10/324). The Beijing genotype was associated with drug-resistant TB (p < 0.0001), including multidrug-resistant TB (p < 0.0001), in our study. An association of the mutation Ser531Leu of the rpoB gene with the Beijing genotype was found (p < 0.0001; OR = 16.0000; 95%CI: 4.9161–52.0740). Among the Beijing isolates, cluster 94-32 showed an association with MDR-TB (p = 0.021). This is why the evaluation of the Beijing genotype and its clusters is needed to control MDR-TB in Kazakhstan.


Introduction
The Beijing genotype is the major component of the East Asian lineage (or lineage 2).Beijing strains were first described in 1995 by van Soolingen et al. [1].Beijing isolates have been found in all continents of the world [2], and they are predominant in Asia (1.4-79.4%)and Eastern Europe (22-71%) [3].Many studies have shown an association between the Beijing genotype and multidrug-resistant tuberculosis (MDR-TB), the most dangerous form of TB that is characterized by resistance to the most effective antibiotics used in TB treatment, rifampicin and isoniazid [4], and its massive spread [3].Beijing MDR isolates were also identified during several TB outbreaks in the US [5] and were the cause of relatively recent outbreaks in some European countries [6,7].
Previous studies have shown that 95% of rifampicin-resistant M. tuberculosis isolates have mutations in the 81bp RRDR (rifampicin-resistance-determining region) of the rpoB The results of the DST revealed the prevalence of drug-resistant clinical isolates (55.6%; 300/540) among the 540 M. tuberculosis samples collected from new TB cases in Kazakhstan.Among the 300 resistant TB forms, MDR-TB associated with the main antibiotics, rifampicin and isoniazid, was identified in 52.3% (157/300) of cases.Four combinations of drug resistance were determined among the MDR samples; 37% out of 52.3% (111/157) of the isolates with MDR displayed resistance to all four first-line antibiotics (rifampicin, isoniazid, ethambutol and streptomycin) that were tested (Table S1).

Evaluation of M. tuberculosis Families
Initially, 24 MIRU-VNTR genotyping was performed for 561 M. tuberculosis clinical isolates.A total of 21 (3.7%) of the isolates showed two bands among the 24 MIRU-VNTR loci, which means a double infection or mixed infection.For this reason, these isolates were further excluded from the study.

Beijing Genotype Clusters in Kazakhstan
The results of the 24 MIRU-VNTR showed that the Beijing family was the most prevalent M. tuberculosis family among the clinical isolates obtained from the new TB cases in Kazakhstan (60%; 324/540).RD (region of difference) analysis revealed that all the 324 Beijing isolates had the RD105 deletion.
The 24 MIRU-VNTR genotyping of the 324 Beijing M. tuberculosis clinical isolates displayed 90 variants of digital profiles.Twenty-three profiles were represented by clusters, which included from 2 to 163 isolates according to the MIRU-VNTRplus nomenclature (Table 1).A total of 79.3% (257/324) of the Beijing isolates were in clusters, while 20.7% (67/324) had unique digital MIRU-VNTR profiles found only in one isolate in our sample collection.A total of 71.2% (183/257) of the clustered isolates of the Beijing genotype showed one of the resistant forms of TB (mono-/poly-resistant and multidrug-resistant TB).The remaining 28.8% (74/257) of the samples were drug-susceptible to the first-line anti-TB drugs that were tested (Table 1).Among the largest Central Asian/Russian cluster 94-32 with the MIRU-VNTR profile 244233352644425153353823, the isolates were mainly resistant to first-line antibiotics (73%; 119/163).The association of cluster 94-32 of the Beijing genotype with drug-resistant forms of TB was not statistically significant (p = 0.149).However, the cluster 94-32 samples were mostly isolated from patients with MDR-TB rather than other drug-resistant TB forms (66.4%; 79/119 vs. 33.6%;40/119) among the antibioticresistant M. tuberculosis samples and showed statistically a significant association (p = 0.021).Among the other Beijing isolates grouped into one group, MDR and mono-/poly-resistant isolates were found almost in equal quantities (50.5%; 53/105 vs. 49.5%;52/105) (Table 2).A comparative analysis of the data also revealed that three other main clusters, 99-32, 94-33 and 100-32, of the Beijing genotype were not associated with infection by drugresistant TB and any form of resistant TB (Table 2).

Determination of Mutations in katG, fabG-inhA, oxyR-ahpC and rpoB Genes of M. tuberculosis Clinical Isolates
The most common mutations in the rpoB (Ser531Leu in codon 531) and the katG (Ser315Thr in codon 315) genes of 540 M. tuberculosis isolates associated with resistance to rifampicin and isoniazid, respectively, were analyzed by allele-specific real-time PCR (AS-RT-PCR).
The results of the AS-RT-PCR displayed that among the rifampicin-resistant isolates (n = 175), the Ser531Leu mutation of rpoB was found in 87.4% (153/175) of the cases.The remaining 12.6% (22/175) resistant isolates did not show the mutation (Table 3).Among the rifampicin-susceptible isolates (n = 365) in 97.8% (357/365) of the cases, no Ser531Leu mutation was determined in the gene.The presence of the Ser531Leu mutation in the rpoB gene was detected among the rest of the eight (2.2%) susceptible isolates.DNA sequencing of the rpoB gene of 22 (12.6%)rifampicin-resistant isolates without Ser531Leu mutation showed mutations in three codons of the rpoB gene (Table 4).Three and two variants of the mutations were determined in codons 526 and 531 among all resistant isolates, respectively.The His526Leu mutation in codon 526 was identified in 5.1% (n = 9) of the cases.Two other mutations were detected in codon 526 of the rpoB gene of two isolates: His526Pro (0.6%; n = 1) and His526Tyr (0.6%; n = 1), respectively.In codon 531, two variants of mutations with amino acid substitutions of serine to phenylalanine Ser531Phe (0.6%; n = 1) and serine to tryptophan Ser531Trp (0.6%; n = 1) were found in two isolates.An amino acid change from leucine to proline was identified in codon 533 (Leu533Pro) of the rpoB gene in two (1.1%) isolates.In 4% (n = 7) of the cases, any mutations in the rpoB gene were not noted.Among 2.2% (n = 8) of the rifampicin-susceptible strains, the Ser531Leu mutation of the rpoB gene was confirmed by Sanger sequencing.The statistical analysis of the obtained data revealed that among the M. tuberculosis rifampicin-resistant clinical isolates with confirmed mutations in different codons of the rpoB gene (n = 177), there was a significant association between the Beijing genotype and the presence of the most common mutation Ser531Leu in codon 531 of the rpoB gene (p < 0.0001; OR = 16.0000;95% CI: 4.9161-52.0740)(Table 5).The statistical analysis of the predominant Central Asian/Russian cluster 94-32 of the Beijing genotype in our sample collection did not show any association with the Ser531Leu mutation of the rpoB gene, as this mutation was detected in almost identical quantities among the isolates of cluster 94-32 and the other Beijing isolates pooled together (97.7% vs. 95.2%;p = 0.4066; OR = 2.1610; 95% CI: 0.3502-13.3347).The comparative analysis of the samples in the other prevalent clusters 100-32, 99-32 and 94-33 with the other Beijing isolates separately did not display an association with Ser531Leu mutation of the rpoB gene (Table 5).
The rifampicin-resistant isolates of the Beijing genotype had a strong association with the Ser531Leu mutation in codon 531 of the rpoB gene (p < 0.0001; OR = 16.0000;95% CI: 4.9161-52.0740).It is interesting to note that the rifampicin-resistant isolates of the LAM (Latin American Mediterranean) genotype had a significant association with the His526Leu mutation in codon 526 of the rpoB gene (p < 0.0001; OR = 141.3333;95% CI: 15.9020-1256.1369).In general, among the non-Beijing group, the LAM genotype revealed a strong correlation with drug-resistant TB (p < 0.0001).
The AS-RT-PCR results showed that the most common mutation with the amino acid change from serine to threonine Ser315Thr in codon 315 of the katG gene was identified in 96.5% (220/228) of the cases among the isoniazid-resistant isolates.The presence of the Ser315Thr mutation in the katG gene was not observed in the other eight (3.5%) isolates (Table 3).Among the 312 isoniazid-susceptible isolates, in 95.8% (299/312) of the cases, the most distributed mutation Ser315Leu of the katG gene was not detected, and in 4.2% of the cases (13/312), the clinical isolates had the mutation.
Further, to identify other mutations in the genes that play an important role in the formation of resistance to isoniazid, Sanger sequencing of the katG, fabG-inhA and oxyR-ahpC genes of eight resistant isolates without the Ser315Thr mutation was carried out.The sequencing results revealed that 0.9% (n = 2) of the samples had a mutation in the fabG-inhA promoter region (15 C-T).The other 2.6% (n = 6) of the isolates showed no mutations in the katG, fabG-inhA and oxyR-ahpC genes (Table 6).The presence of the Ser315Thr mutation in the katG gene of 13 isoniazid-susceptible isolates was confirmed by Sanger sequencing.Sequencing of the fabG-inhA and oxyR-ahpC promoter regions of these 13 isoniazid-susceptible samples did not display any changes.The comparative analysis showed that among the isoniazid-resistant isolates with confirmed mutations in the katG or the fabG-inhA genes (n = 235), over 97% of the samples had the Ser315Thr mutation in the katG gene, regardless of whether they belonged to the Beijing genotype or non-Beijing genotypes of M. tuberculosis (p = 0.2603; OR = 4.9744; 95% CI: 0.3046-81.2405).In terms of the Beijing genotype clusters (n = 195), a similar trend was identified to that described above.Clusters 94-32, 100-32, 99-32 and 94-33, in comparison with the other Beijing isolates individually, did not have an association with the main mutation Ser315Thr in codon 315 of the katG gene, as almost an equal number of samples in all the cluster groups had this mutation (Table 5).

Discussion
Distribution of drug-resistant TB forms, including rifampicin-resistant TB (RR-TB), that is resistant to the main first-line antibiotic rifampicin and MDR-TB (resistant to rifampicin and isoniazid), the most dangerous form of TB remains one of the critical issues of the healthcare system in Kazakhstan.According to a World Health Organization (WHO) report, Kazakhstan is on the list of 30 countries with high rates of MDR/RR-TB.In 2021, 3.1% more cases of MDR/RR-TB (n = 450,000) were registered in the world compared to 2020, and >50% of the MDR/RR-TB cases among previously treated patients were detected from FSU countries [28].Therefore, it is important to investigate the most distributed M. tuberculosis genotypes and their clusters in Kazakhstan as well as to monitor their spread and apply adequate control measures to combat tuberculosis.
The prevalence of the Beijing family isolates among M. tuberculosis samples from Kazakhstan was also noted in earlier studies.The Beijing genotype was found in 65.3% (177/271) of cases among isolates from three regions of Kazakhstan (South and North Kazakhstan and Almaty city) [22].Among the isolates resistant and susceptible to pyrazinamide, Beijing family strains were identified in 78.4% (58/74) of the cases [20].In a study by Skiba et al., 72.2% (109/151) of the isolates belonged to the Beijing family, and 101 out of 109 Beijing isolates were grouped as 94-32 and related types [23].In a study by Hillemann et al., the Beijing genotype was identified in 64.1% (59/92) of cases among MDR isolates and in 64% (32/50) of cases among isoniazid-but not rifampicin-resistant (INH r /RMP s ) strains [21].The study revealed an association of the Ser531Leu mutation of the rpoB gene with the Beijing genotype among the MDR group (p = 0.027) and an association of the Ser315Thr mutation of the katG gene among the INH r /RMP s group (p = 0.012).In the present study, the Beijing genotype clusters distributed in Kazakhstan were described, their drug susceptibility profiles and correlation with the main mutations Ser531Leu in the rpoB gene and Ser315Thr in the katG gene associated with resistance to the most effective first-line antibiotics, rifampicin and isoniazid, respectively, were investigated.
In our study, the Beijing genotype was associated with drug-resistant TB compared to drug-susceptible TB (p < 0.0001).Among the drug-resistant M. tuberculosis clinical isolates, the Beijing genotype had a significant association with MDR-TB compared to TB with other DR (mono-/poly-resistance) (p < 0.0001).In previous studies conducted in Kazakhstan, the Beijing genotype also showed an association with infection by drug-resistant TB, including MDR-TB [23].
In our research, drug-resistant TB slightly prevailed among the isolates of the Central Asian/Russian cluster 94-32 compared to the other Beijing isolates pooled together (73% vs. 65.2%;p = 0.149).However, the Beijing cluster 94-32 was found almost two times more among MDR-TB patients than TB patients with other DR (66.4% vs. 33.6%;p = 0.021).Other Beijing clusters, including the prevalent Beijing clusters (94-33, 100-32 and 99-32) found in this study, did not correlate with drug-resistant TB and MDR-TB.In the earlier study by Skiba et al. [23], association of MDR-TB with cluster 94-32 was not confirmed (p = 0.07).
The Central Asian/Russian type 94-32 is highly dominant in Central Asian countries [31] and the Russian Federation [16].The Russian cluster 100-32 is associated with MDR-TB and is widely distributed in different regions of Russia [27].The cluster 100-32 is one of the predominant clusters found in Central Asia and Eastern Europe [3,31].According to a report from the European Center for Disease Prevention and Control (2016), cluster 94-32 was determined among 23.7% of MDR-TB patients from 14 countries in Europe, and the Russian cluster 100-32 was detected among 33.6% of individuals with MDR-TB from 11 European countries [17].The Beijing 94-32 and 100-32 clusters were also identified in immigrants from FSU countries in the US [16].Among Central Asian countries (Kyrgyzstan, Tajikistan and Uzbekistan), the Beijing cluster 94-32 had an identical drug resistance profile as other Beijing isolates, while the Beijing cluster 100-32, in 99.3% (66/70) of cases, was MDR, pre-XDR or XDR [31].The Beijing clusters 94-33 and 99-32 were found in FSU countries [32,33].
In our study, 4.1% (22/540) of the clinical isolates were classified as 'unknown', as they were not linked to any known M. tuberculosis families.Isolates with 'undefined' families were also found in other investigations of Central Asian isolates in 0.7-5.3% of cases [23,31].
Among the rifampicin-resistant M. tuberculosis isolates (n = 175), mutations in three codons of the rpoB gene were identified in 96% (168/175) of the cases.The main part of the resistant isolates (87.4%; 153/175) revealed the most commonly reported Ser531Leu mutation of the rpoB gene.This Ser531Leu mutation was significantly associated with the Beijing genotype in our study (p < 0.0001; OR = 16.0000;95%CI: 4.9161-52.0740).An association of the Central Asian/Russian cluster 94-32 and three other dominant Beijing clusters (94-33, 100-32 and 99-32) with the Ser531Leu mutation of the rpoB gene was not found.
LAM genotype among the non-Beijing isolates was associated with drug resistance (p < 0.0001).Among the rifampicin-resistant isolates, the LAM genotype was associated with the His526Leu mutation of the rpoB gene (p < 0.0001; OR = 141.3333;95%CI: 15.9020-1256.1369).This mutation of the rpoB gene was also identified in the genomes of drug-resistant LAM isolates (66.7%; 2/3) from Kazakhstan [34].
Among the isoniazid-resistant clinical isolates (n = 228), mutations in the katG and the fabG-inhA genes were determined in 97.4% (222/228) of the cases.The majority of the samples (96.5%; 220/228) had the most common Ser315Thr mutation in codon 315 of the katG gene.The association of the Ser315Thr mutation of the katG gene with the Beijing genotype and its clusters were not observed in our research.
The Ser531Leu mutation of the rpoB gene was associated with the Beijing genotype among MDR-TB (p = 0.027) cases in Kazakhstan in an earlier study [21], and the Ser315Thr mutation of the katG gene was shown to have an association with isoniazid-resistant (but rifampicin-susceptible) Beijing isolates (p = 0.012).However, in East Asian countries, the number of isolates with the most prevalent mutations, Ser315Thr in the katG gene and Ser531Leu in the rpoB gene, were similar between Beijing and non-Beijing groups [35].
In 4% (7/175) and 2.6% (6/228) of the cases, the rifampicin-and isoniazid-resistant clinical isolates did not reveal any mutations in the rpoB and katG, fabG-inhA, oxyR-ahpC genes, respectively.The absence of mutations in specific drug-resistant genes has been recorded in 2.3-10.38% of cases in various studies [36][37][38][39].Mutations in other genes may cause resistance to ant i-TB drugs in these resistant isolates.For example, whole-genome sequencing data of M. tuberculosis isolates from high TB-burden countries showed 23 novel katG mutations in isoniazid-resistant isolates [40] and mutations outside of the rpoB gene in rifampicin-resistant isolates [41,42].Yet, genomic data of MDR, pre-XDR and XDR strains from Kazakhstan identified new genetic variants that may play an important role in the formation of drug resistance [24,25,41].
In addition, 2.2% (8/365) of the rifampicin-and 4.2% (13/312) of the isoniazidsusceptible isolates in this work showed the most common mutations, Ser531Leu of the rpoB gene and Ser315Thr of the katG gene, respectively.These isolates may have a low resistance to rifampicin and isoniazid [43].

Clinical Isolates of M. tuberculosis and DST
Clinical isolates of M. tuberculosis (n = 561) were collected from new cases of pulmonary tuberculosis from different oblasts of Kazakhstan.All the microbiological investigations, including the identification and isolation of M. tuberculosis cultures from the sputum of TB patients, were performed at the National Reference Laboratory of the National Scientific Center of Phthisiopulmonology (Almaty city).Drug susceptibility testing (DST) was carried out for four first-line anti-TB drugs by a microbiological method using a BACTEC-Mycobacterial Growth Indicator Tube (MGIT) 960 (BD Diagnostic System, Franklin Lakes, NJ, USA) with critical drug concentrations of 0.5 µg/mL for rifampicin, 0.1 µg/mL for isoniazid, 5 µg/mL for ethambutol and 1 µg/mL for streptomycin.

Genotyping
DNA of M. tuberculosis samples was isolated according to a standard protocol as described earlier [44].
The 24 MIRU-VNTR genotyping was performed for the M. tuberculosis clinical isolates as shown previously [22,45] to identify Beijing and non-Beijing M. tuberculosis isolates.The obtained MIRU-VNTR profiles were analyzed using the www.miru-vntrplus.orgweb resource (https://www.miru-vntrplus.org/MIRU/index.faces,accessed on 1 June 2023), where MLVA types (clusters) were assigned to the isolates.Two or more isolates with identical MIRU-VNTR profiles in a phylogenetic group were considered as a cluster [45].
The Beijing family isolates were additionally identified by RD analysis.Determination of RD105 deletion was conducted as shown previously [29,46].Typing was carried out by real-time PCR using SYBR green PCR master mix (Applied Biosystems, Waltham, MA, USA) in a CFX96 Real-time System (Bio-Rad, Hercules, CA, USA) with an initial denaturation step of 95 • C for 3 min, followed by 41 cycles of denaturation at 95 • C for 5 s, annealing at 60 • C for 8 s and elongation at 72 • C for 10 s.Data acquisition was carried out on the SYBR channel, and melting curves of the amplification products were obtained from 72 • C to 97 • C (in 0.5 • C increments).

Detection of Mutations in Drug-Resistant Genes
Allele-specific real-time PCR (AS-RT-PCR) was performed for 540 clinical isolates for screening of the most distributed mutations, Ser531Leu in codon 531 of the rpoB gene and Ser315Thr in codon 315 of the katG gene, associated with drug resistance to rifampicin and isoniazid, respectively.AS-RT-PCR was carried out using SYBR green PCR master mix (Applied Biosystems) with similar PCR conditions to those used for the RD105 locus.The PCR reaction was performed with the allele-specific and reference primers (Table S2).The following isolates with confirmed by the Sanger sequencing results were used as controls: 2 rifampicin-resistant isolates with the Ser531Leu mutation and 2 isoniazid-resistant isolates with the Ser315Thr mutation as well as 2 rifampicin-susceptible and 2 isoniazid-susceptible isolates.Sterile mQ water was used as a negative control.

Statistical Analysis
IBM SPSS Statistics 24.0 and Medcalc online [48] were used to carry out the statistical analysis of the obtained data.The chi-squared test (χ 2 ) and Fisher's exact test were used to detect any significant differences between the two groups.Yates' corrected χ 2 and p-values were calculated with a 95% confidence interval (CI) for the mean where necessary.The results were considered statistically significant if the p-value was equal to or less than 0.05.

Conclusions
The Beijing genotype is the main M. tuberculosis genotype spread in Kazakhstan.The genotype correlates with the presence of the Ser531Leu mutation in codon 531 of the rpoB gene, which is associated with resistance to the most effective first-line anti-TB drug, rifampicin.Clusters 94-32, 94-33, 100-32 and 99-32 were the predominant clusters of the Beijing genotype among the new TB cases.The data obtained from this study revealed the essential role of the Beijing genotype and its Central Asian/Russian cluster 94-32 in the distribution of drug-resistant TB, particularly MDR-TB, in Kazakhstan.Therefore, necessary measures need to be taken to combat this epidemic.It is important to pay attention to the investigation and control of the Beijing genotype and its clusters for the monitoring of drug-resistant, especially multidrug-resistant, M. tuberculosis circulating in Kazakhstan and Central Asian countries.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antibiotics13010009/s1,Table S1: Description of M. tuberculosis clinical isolates resistant to antituberculosis drugs; Table S2: Sequences of primers used in AS-RT-PCR to determine resistance in the analyzed M. tuberculosis genes.

Table 1 .
Drug susceptibility of Beijing genotype clusters (two or more isolates) among new TB cases.

Table 2 .
Association analysis of the relationship between different M. tuberculosis genotypes, includingBeijing isolates with drug resistance among new TB cases.
* Statistically significant result; these values were not calculated in SPSS.

Table 3 .
Determination of Ser531Leu mutation in the rpoB gene and Ser315Thr mutation in the katG gene of clinical isolates of M. tuberculosis.

Table 4 .
Variants of amino acid substitutions in the rpoB gene of clinical isolates of M. tuberculosis.

Table 5 .
Statistical analysis of the association between different M. tuberculosis genotypes, includingBeijing isolates with mutations in the rpoB and the katG genes among new TB cases.
* Statistically significant result; these values were not calculated in SPSS.

Table 6 .
Mutations in genes of M. tuberculosis clinical isolates associated with resistance to isoniazid.